Pathway: WNT ligand biogenesis and trafficking
Reactions in pathway: WNT ligand biogenesis and trafficking :
WNT ligand biogenesis and trafficking
19 WNT proteins have been identified in human cells. The WNTs are members of a conserved metazoan family of secreted morphogens that activate several signaling pathways in the responding cell: the canonical (beta-catenin) WNT signaling cascade and several non-canonical pathways, including the planar cell polarity (PCP), the regulation of intracellular calcium signaling and activation of JNK kinases. WNT proteins exist in a gradient outside the secreting cell and are able to act over both short and long ranges to promote proliferation, changes in cell migration and polarity and tissue homeostasis, among others (reviewed in Saito-Diaz et al, 2012; Willert and Nusse, 2012).
The WNTs are ~40kDa proteins with 23 conserved cysteine residues in the N-terminal that may form intramolecular disulphide bonds. They also contain an N-terminal signal sequence and a number of N-linked glycosylation sites (Janda et al, 2012). In addition to being glycosylated, WNTs are also lipid-modified in the endoplasmic reticulum by a WNT-specific O-acyl-transferase, Porcupine (PORCN), contributing to their characteristic hydrophobicity. PORCN-dependent palmitoylation is required for the secretion of WNT as well as its signaling activity, as either depletion of PORCN or mutation of the conserved serine acylation site results in the intracellular accumulation of WNT ligand (Takada et al, 2006; Barrott et al, 2011; Biechele et al, 2011; reviewed in Willert and Nusse, 2012).
Secretion of WNT requires a number of other dedicated factors including the sorting receptor Wntless (WLS) (also knownas Evi, Sprinter, and GPR177), which binds WNT and escorts it to the cell surface (Banziger et al, 2006; Bartscherer et al, 2006; Goodman et al, 2006). A WNT-specific retromer containing SNX3 is subsequently required for the recycling of WLS back to the Golgi (reviewed in Herr et al, 2012; Johannes and Wunder, 2011). Once at the cell surface, WNT makes extensive contacts with components of the extracellular matrix such as heparan sulphate proteoglycans (HSPGs) and may be bound by any of a number of regulatory proteins, including WIFs and SFRPs. The diffusion of the WNT ligand may be aided by its packing either into WNT multimers, exosomes or onto lipoprotein particles to shield the hydrophobic lipid adducts from the aqueous extracellular environment (Gross et al, 2012; Luga et al, 2012, Korkut et al, 2009; reviewed in Willert and Nusse, 2012).
The WNTs are ~40kDa proteins with 23 conserved cysteine residues in the N-terminal that may form intramolecular disulphide bonds. They also contain an N-terminal signal sequence and a number of N-linked glycosylation sites (Janda et al, 2012). In addition to being glycosylated, WNTs are also lipid-modified in the endoplasmic reticulum by a WNT-specific O-acyl-transferase, Porcupine (PORCN), contributing to their characteristic hydrophobicity. PORCN-dependent palmitoylation is required for the secretion of WNT as well as its signaling activity, as either depletion of PORCN or mutation of the conserved serine acylation site results in the intracellular accumulation of WNT ligand (Takada et al, 2006; Barrott et al, 2011; Biechele et al, 2011; reviewed in Willert and Nusse, 2012).
Secretion of WNT requires a number of other dedicated factors including the sorting receptor Wntless (WLS) (also knownas Evi, Sprinter, and GPR177), which binds WNT and escorts it to the cell surface (Banziger et al, 2006; Bartscherer et al, 2006; Goodman et al, 2006). A WNT-specific retromer containing SNX3 is subsequently required for the recycling of WLS back to the Golgi (reviewed in Herr et al, 2012; Johannes and Wunder, 2011). Once at the cell surface, WNT makes extensive contacts with components of the extracellular matrix such as heparan sulphate proteoglycans (HSPGs) and may be bound by any of a number of regulatory proteins, including WIFs and SFRPs. The diffusion of the WNT ligand may be aided by its packing either into WNT multimers, exosomes or onto lipoprotein particles to shield the hydrophobic lipid adducts from the aqueous extracellular environment (Gross et al, 2012; Luga et al, 2012, Korkut et al, 2009; reviewed in Willert and Nusse, 2012).
WNT signaling pathways control a wide range of developmental and adult process in metozoans including cell proliferation, cell fate decisions, cell polarity and stem cell maintenance (reviewed in Saito-Diaz et al, 2013; MacDonald et al, 2009). The pathway is named for the WNT ligands, a large family of secreted cysteine-rich glycoproteins. At least 19 WNT members have been identified in humans and mice with distinct expression patterns during development (reviewed in Willert and Nusse, 2012). These ligands can activate at least three different downstream signaling cascades depending on which receptors they engage.
In the so-called 'canonical' WNT signaling pathway, WNT ligands bind one of the 10 human Frizzled (FZD) receptors in conjunction with the LRP5/6 co-receptors to activate a transcriptional cascade that controls processes such as cell fate, proliferation and self-renenwal of stem cells. Engagement of the FZD-LRP receptor by WNT ligand results in the stabilization and translocation of cytosolic beta-catenin to the nucleus where it is a co-activator for LEF (lymphoid enhancer-binding factor)- and TCF (T cell factor) -dependent transcription. In the absence of WNT ligand, cytosolic beta-catenin is phosphorylated by a degradation complex consisting of glycogen synthase kinase 3 (GSK3), casein kinase 1 (CK1), Axin and Adenomatous polyposis coli (APC), and subsequently ubiquitinated and degraded by the 26S proteasome (reviewed in Saito-Diaz et al, 2013; Kimmelman and Xu, 2006).
In addition to the beta-catenin-dependent transcriptional response, WNT signaling can also activate distinct non-transcriptional pathways that regulate cell migration and polarity. These beta-catenin-independent 'non-canonical' pathways signal through Frizzled receptors independently of LRP5/6, or occur through the tyrosine kinase receptors ROR and RYK (reviewed in Veeman et al, 2003; James et al, 2009). Non-canonical WNT pathways are best studied in Drosophila where the planar cell polarity (PCP) pathway controls the orientation of wing hairs and eye facets, but are also involved in processes such as convergent extension, neural tube closure, inner ear development and hair orientation in vertebrates and mammals(reviewed in Seifert and Mlodzik, 2007; Simons and Mlodzik, 2008). In the PCP pathway, binding of WNT ligand to the FZD receptor leads to activation of small Rho GTPases and JNK, which regulate the cytoskeleton and coordinate cell migration and polarity (reviewed in Lai et al, 2009; Schlessinger et al, 2009). In some cases, a FZD-WNT interaction increases intracellular calcium concentration and activates CaMK II and PKC; this WNT calcium pathway promotes cell migration and inhibits the canonical beta-catenin dependent transcriptional pathway (reviewed in Kuhl et al, 2000; Kohn and Moon, 2005; Rao et al 2010). Binding of WNT to ROR or RYK receptors also regulates cell migration, apparently through activation of JNK or SRC kinases, respectively, however the details of these pathways remain to be worked out (reviewed in Minami et al, 2010).
Although the WNT signaling pathways were originally viewed as discrete, linear pathways controlled by defined subsets of 'canonical' or 'non-canonical' ligands and receptors, the emerging evidence is challenging this notion. Instead, the specificity and the downstream response appear to depend on the particular cellular context and vary with species, tissue and stage of development (reviewed in van Amerongen and Nusse, 2009; Rao et al, 2010).
In the so-called 'canonical' WNT signaling pathway, WNT ligands bind one of the 10 human Frizzled (FZD) receptors in conjunction with the LRP5/6 co-receptors to activate a transcriptional cascade that controls processes such as cell fate, proliferation and self-renenwal of stem cells. Engagement of the FZD-LRP receptor by WNT ligand results in the stabilization and translocation of cytosolic beta-catenin to the nucleus where it is a co-activator for LEF (lymphoid enhancer-binding factor)- and TCF (T cell factor) -dependent transcription. In the absence of WNT ligand, cytosolic beta-catenin is phosphorylated by a degradation complex consisting of glycogen synthase kinase 3 (GSK3), casein kinase 1 (CK1), Axin and Adenomatous polyposis coli (APC), and subsequently ubiquitinated and degraded by the 26S proteasome (reviewed in Saito-Diaz et al, 2013; Kimmelman and Xu, 2006).
In addition to the beta-catenin-dependent transcriptional response, WNT signaling can also activate distinct non-transcriptional pathways that regulate cell migration and polarity. These beta-catenin-independent 'non-canonical' pathways signal through Frizzled receptors independently of LRP5/6, or occur through the tyrosine kinase receptors ROR and RYK (reviewed in Veeman et al, 2003; James et al, 2009). Non-canonical WNT pathways are best studied in Drosophila where the planar cell polarity (PCP) pathway controls the orientation of wing hairs and eye facets, but are also involved in processes such as convergent extension, neural tube closure, inner ear development and hair orientation in vertebrates and mammals(reviewed in Seifert and Mlodzik, 2007; Simons and Mlodzik, 2008). In the PCP pathway, binding of WNT ligand to the FZD receptor leads to activation of small Rho GTPases and JNK, which regulate the cytoskeleton and coordinate cell migration and polarity (reviewed in Lai et al, 2009; Schlessinger et al, 2009). In some cases, a FZD-WNT interaction increases intracellular calcium concentration and activates CaMK II and PKC; this WNT calcium pathway promotes cell migration and inhibits the canonical beta-catenin dependent transcriptional pathway (reviewed in Kuhl et al, 2000; Kohn and Moon, 2005; Rao et al 2010). Binding of WNT to ROR or RYK receptors also regulates cell migration, apparently through activation of JNK or SRC kinases, respectively, however the details of these pathways remain to be worked out (reviewed in Minami et al, 2010).
Although the WNT signaling pathways were originally viewed as discrete, linear pathways controlled by defined subsets of 'canonical' or 'non-canonical' ligands and receptors, the emerging evidence is challenging this notion. Instead, the specificity and the downstream response appear to depend on the particular cellular context and vary with species, tissue and stage of development (reviewed in van Amerongen and Nusse, 2009; Rao et al, 2010).
Signal transduction is a process in which extracellular signals elicit changes in cell state and activity. Transmembrane receptors sense changes in the cellular environment by binding ligands, such as hormones and growth factors, or reacting to other types of stimuli, such as light. Stimulation of transmembrane receptors leads to their conformational change which propagates the signal to the intracellular environment by activating downstream signaling cascades. Depending on the cellular context, this may impact cellular proliferation, differentiation, and survival. On the organism level, signal transduction regulates overall growth and behavior.
Receptor tyrosine kinases (RTKs) transmit extracellular signals by phosphorylating their protein partners on conserved tyrosine residues. Some of the best studied RTKs are EGFR (reviewed in Avraham and Yarden, 2011), FGFR (reviewed in Eswarakumar et al, 2005), insulin receptor (reviewed in Saltiel and Kahn, 2001), NGF (reviewed in Reichardt, 2006), PDGF (reviewed in Andrae et al, 2008) and VEGF (reviewed in Xie et al, 2004). RTKs frequently activate downstream signaling through RAF/MAP kinases (reviewed in McKay and Morrison, 2007 and Wellbrock et al 2004), AKT (reviewed in Manning and Cantley, 2007) and PLC- gamma (reviewed in Patterson et al, 2005), which ultimately results in changes in gene expression and cellular metabolism.
Receptor serine/threonine kinases of the TGF-beta family, such as TGF-beta receptors (reviewed in Kang et al. 2009) and BMP receptors (reviewed in Miyazono et al. 2009), transmit extracellular signals by phosphorylating regulatory SMAD proteins on conserved serine and threonine residues. This leads to formation of complexes of regulatory SMADs and SMAD4, which translocate to the nucleus where they act as transcription factors.
WNT receptors transmit their signal through beta-catenin. In the absence of ligand, beta-catenin is constitutively degraded in a ubiquitin-dependent manner. WNT receptor stimulation releases beta-catenin from the destruction complex, allowing it to translocate to the nucleus where it acts as a transcriptional regulator (reviewed in MacDonald et al, 2009 and Angers and Moon, 2009). WNT receptors were originally classified as G-protein coupled receptors (GPCRs). Although they are structurally related, GPCRs primarily transmit their signals through G-proteins, which are trimers of alpha, beta and gamma subunits. When a GPCR is activated, it acts as a guanine nucleotide exchange factor, catalyzing GDP to GTP exchange on the G-alpha subunit of the G protein and its dissociation from the gamma-beta heterodimer. The G-alpha subunit regulates the activity of adenylate cyclase, while the gamma-beta heterodimer can activate AKT and PLC signaling (reviewed in Rosenbaum et al. 2009, Oldham and Hamm 2008, Ritter and Hall 2009).
NOTCH receptors are activated by transmembrane ligands expressed on neighboring cells, which results in cleavage of NOTCH receptor and release of its intracellular domain. NOTCH intracellular domain translocates to the nucleus where it acts as a transcription factor (reviewed in Kopan and Ilagan, 2009).
Integrins are activated by extracellular matrix components, such as fibronectin and collagen, leading to conformational change and clustering of integrins on the cell surface. This results in activation of integrin-linked kinase and other cytosolic kinases and, in co-operation with RTK signaling, regulates survival, proliferation and cell shape and adhesion (reviewed in Hehlgans et al, 2007) .
Besides inducing changes in gene expression and cellular metabolism, extracellular signals that trigger the activation of Rho GTP-ases can trigger changes in the organization of cytoskeleton, thereby regulating cell polarity and cell-cell junctions (reviewed in Citi et al, 2011).
Receptor tyrosine kinases (RTKs) transmit extracellular signals by phosphorylating their protein partners on conserved tyrosine residues. Some of the best studied RTKs are EGFR (reviewed in Avraham and Yarden, 2011), FGFR (reviewed in Eswarakumar et al, 2005), insulin receptor (reviewed in Saltiel and Kahn, 2001), NGF (reviewed in Reichardt, 2006), PDGF (reviewed in Andrae et al, 2008) and VEGF (reviewed in Xie et al, 2004). RTKs frequently activate downstream signaling through RAF/MAP kinases (reviewed in McKay and Morrison, 2007 and Wellbrock et al 2004), AKT (reviewed in Manning and Cantley, 2007) and PLC- gamma (reviewed in Patterson et al, 2005), which ultimately results in changes in gene expression and cellular metabolism.
Receptor serine/threonine kinases of the TGF-beta family, such as TGF-beta receptors (reviewed in Kang et al. 2009) and BMP receptors (reviewed in Miyazono et al. 2009), transmit extracellular signals by phosphorylating regulatory SMAD proteins on conserved serine and threonine residues. This leads to formation of complexes of regulatory SMADs and SMAD4, which translocate to the nucleus where they act as transcription factors.
WNT receptors transmit their signal through beta-catenin. In the absence of ligand, beta-catenin is constitutively degraded in a ubiquitin-dependent manner. WNT receptor stimulation releases beta-catenin from the destruction complex, allowing it to translocate to the nucleus where it acts as a transcriptional regulator (reviewed in MacDonald et al, 2009 and Angers and Moon, 2009). WNT receptors were originally classified as G-protein coupled receptors (GPCRs). Although they are structurally related, GPCRs primarily transmit their signals through G-proteins, which are trimers of alpha, beta and gamma subunits. When a GPCR is activated, it acts as a guanine nucleotide exchange factor, catalyzing GDP to GTP exchange on the G-alpha subunit of the G protein and its dissociation from the gamma-beta heterodimer. The G-alpha subunit regulates the activity of adenylate cyclase, while the gamma-beta heterodimer can activate AKT and PLC signaling (reviewed in Rosenbaum et al. 2009, Oldham and Hamm 2008, Ritter and Hall 2009).
NOTCH receptors are activated by transmembrane ligands expressed on neighboring cells, which results in cleavage of NOTCH receptor and release of its intracellular domain. NOTCH intracellular domain translocates to the nucleus where it acts as a transcription factor (reviewed in Kopan and Ilagan, 2009).
Integrins are activated by extracellular matrix components, such as fibronectin and collagen, leading to conformational change and clustering of integrins on the cell surface. This results in activation of integrin-linked kinase and other cytosolic kinases and, in co-operation with RTK signaling, regulates survival, proliferation and cell shape and adhesion (reviewed in Hehlgans et al, 2007) .
Besides inducing changes in gene expression and cellular metabolism, extracellular signals that trigger the activation of Rho GTP-ases can trigger changes in the organization of cytoskeleton, thereby regulating cell polarity and cell-cell junctions (reviewed in Citi et al, 2011).